| Abstract: | Withaferin A (WA) is one of the major bioactive steroidal lactones with extensive pharmacological activities present in the plant Withania somnifera. The absolute oral bioavailability of WA remains unknown and human‐related in vitro data are not available. Therefore, in the present study, the absolute oral bioavailability of WA in male rats and the in vitro screening of absorption factors by Q‐trap and LC–MS/MS analysis were conducted to explore possible clinical properties of WA. The developed and validated analytical methods were successfully applied to the pharmacokinetic studies and in vitro measurement of WA. The oral bioavailability was determined to be 32.4 ± 4.8% based on intravenous (5 mg/kg) and oral (10 mg/kg) administrations of WA in male rats. The in vitro results showed that WA could be easily transported across Caco‐2 cells and WA did not show as a substrate for P‐glycoprotein. Moreover, the stability of WA was similar between male rat and human in simulated gastric fluid (stable), in intestinal microflora solution (slow decrease) and in liver microsomes (rapid depletion, with a half‐life of 5.6 min). As such, the first‐pass metabolism of WA was further verified by rat intestine‐liver in situ perfusion, revealing that WA rapidly decreased and 27.1% remained within 1 h, while the content of three major metabolites (M1, M4, M5) identified by Q‐trap increased. This perfusion result is consistent with the oral bioavailability results in vivo. The first‐pass metabolism of WA might be the main barrier in achieving good oral bioavailability in male rats and it is predicted to be similar in humans. This study may hold clinical significance. |
